Cerebral edema (CE, or brain swelling) is a major contributor to the damage caused by stroke, traumatic brain injury, and bacterial meningitis. Stroke alone is the third leading cause of death in the USA and the leading cause of disability. Unfortunately, few treatment options are available for CE, and these are of limited efficacy. Central in the development of CE is the H2O channel aquaporin-4 (AQP4) in the astrocytic endfeet that envelope CNS blood vessels at the blood-brain barrier (BBB). AQP4-null mice do substantially better than their wild-type counterparts in experimental models of ischemic stroke, retinal ischemia, and bacterial meningitis. In the past, many targets of CE have failed to produce effective therapeutics. However, because AQP4 is the key gatekeeper for H2O uptake across the BBB, it is a promising new drug target, supported by a genetic proof-of-principle. A unique aspect of the present proposal is the recognition that the AQPs are bifunctional proteins that are permeable not only to H2O, but also to dissolved gases (CO2, O2, NO). Although developing a 1st generation drug for such a novel target entails risk, the potential benefits of an effective and noninvasive treatment for cerebral edema are far greater. In the words of Dr. George Richerson (Chief of Neurology at the University of Iowa), "As a neurologist, I really can't think of a drug that would have more of an impact in neurology than one that treated cerebral edema." The long-term goal of Aeromics is to develop a drug that treats cerebral edema by blocking the water permeability of AQP4, leaving gas permeability intact. In its first Phase I SBIR (NIH/NINDS 1R43NS060199), Aeromics (a) developed a successful high-throughput assay and appropriate counter screens, and (b) tested this assay in a 21k-compound pilot screen. This pilot screen identified 6 hits from four different structural classes. These hits are currently being developed as part of an ongoing Phase II program (NIH/NINDS 2R44NS060199). Our goal for this new application is to scale-up from pilot screen to a high-throughput screen (HTS) with a non- overlapping group of 100k compounds to explore a wider chemical space, thereby ensuring that a sufficient number of leads transition through the downstream elements of our drug-discovery program, minimizing overall program risk. We will: (1) perform a 100k-compound HTS at the University of Cincinnati's Drug Discovery Center, identifying more novel structural classes that block AQP4, (2) counter screen using a sensitive cell-based light scattering assay (developed in part at Aeromics under NIH/NINDS 1R43NS060199), (3) test hits for direct interaction with AQP4 (AQP4 proteoliposomes-shrinking assay), and (4) verify these results by cell volume cytometry. Finally, we will evaluate each hit across a panel of aquaporin family members to ascertain specificity. The proposed work will prepare Aeromics for a new Phase II SBIR that will include medicinal chemistry and a pharmacological proof-of-concept, and will ensure the successful transition of new drug candidates from preclinical work to filing an IND application for treating stroke-induced cerebral edema. PUBLIC HEALTH RELEVANCE: Stroke is the third leading cause of death in the United States, resulting in 137,000 annual fatalities. Many stroke survivors are disabled, leading to a combined annual medical cost of ~$70 billion. Brain swelling (cerebral edema, CE) is a major contributor to the damage caused by stroke, traumatic brain injury (TBI), and bacterial meningitis. Unfortunately, only a few treatment options are available for CE, and these are of limited efficacy. Recent studies have shown that the protein AQP4 is the primary route for water uptake by the brain, making it a promising new target for CE. In a previous Phase I SBIR, Aeromics developed technologies to identify drug candidates for treating CE through inhibition of AQP4, and discovered 6 new compounds in a pilot program. These compounds are currently under development in a Phase II SBIR. With the current screening technologies in place, we are ready to scale-up our drug discovery program to complete a more thorough search for new drug candidates.